8-Azido-dATP, a photoaffinity analog of dATP, is an efficient nucleotide sub-trate for TDTase with a Km of 53 μM (when the Km for dATP is 140 μM) (16). In preparing a series of PCR reactions, the four dNTPs can be prepared as a master mix, an aliquot of which is added to each PCR reaction in the series. An elevated level of S-cdG has been detected in Xeroderma pigmentosum, Cockayne syndrome, breast cancer patients, and aged mice. Because the tubes/plates are not opened, there is minimal risk of contamination by PCR amplicons. Two of the binding sites do not contain nucleotides.
The nonisotopically labeled oligonucleotides (or primers) are suitable for use in nucleic acid hybridization, DNA sequencing, and PCR priming. The labeling of the second cDNA strand with uridines requires the removal of dTTP from the reaction. Gel electrophoretic analysis or “melt-curve analysis” can confirm the presence of appropriate and spurious amplicons. Following primer-extension, the enzyme-product complex was crystallized and cocrystal structures of Bst DNAP-I were solved to resolutions of 1.5 – … It can detect a variable level of a minority component at least to 1%. The crystal structure of the hexameric gp4 reveals the presence of three different conformational states adopted by the nucleotide-binding site within the protein's ring. We envision the following mechanism for coupling of nucleotide hydrolysis to translocation of gp4 on ssDNA. In contrast to other replicative helicases primarily hydrolyzing ATP, gp4 uses dTTP  to fuel its reactions. 8.
Hydrolysis probes can be used for measuring gene expression and for genotyping. In addition to PCR primers, each assay has two probes: a long oligonucleotide, the “Anchor Probe” and a short oligonucleotide, the “Sensor Probe”. In the Dpo4:DNA( S-cdG):dTTP structure, S-cdG induced a loop structure and caused an unusual 5′-template base clustering at the active site, providing the first structural evidence of the previously suggested template loop structure that can be induced by a cyclopurine lesion. The 560 nucleotides represent one copy of the target sequence.
Experiments in which the wild-type gp4 were mixed in different ratios with catalytically inactive variant of gp4 harboring a single amino-acid substitution of glutamine 343 to glutamic acid (Fig.
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